Potentiometric biosensors are known. Conover et al., U.S. Pat. No. 4,713,165 (issued Dec. 15, 1987), discloses a sensor having ion selective electrodes. The sensor includes three cells, each cell having a reference half-cell (including a reference electrode and a reference fluid) and a measuring half-cell. The half-cells are separated by an ion selective membrane, and each half-cell is connected to an adjacent measuring half-cell by a porous material that permits ionic flow. The measuring half-cell may further include a membrane which includes an enzyme, an enzyme substrate, or an antigen. Generally, a sample that contains an analyte of interest is added to a measuring half-cell. A reaction sequence is set up to generate some ion (for example, ammonium ion or hydronium ion). Therefore, the activity of the ion in the measuring half-cell changes. The change in potential of the measuring half-cell versus the reference half-cell reflects the change in activity in the measuring half-cell.
When a reference solution and a calibrating solution are added to the other measuring half-cells, the change in potential in the sample-containing half-cell may be correlated to the amount of analyte in the sample.
Schiller et al., U.S. Pat. No. 4,340,448 (issued Jul. 20, 1982), discloses a potentiometric device for the assay of an analyte. The device has a working electrode and a reference electrode. An oxidase enzyme is immobilized on the working electrode. The working electrode must be made of a material, such as platinum, capable of measuring potential as a function of the change in the concentration of hydrogen peroxide. For example, a sample containing glucose may be added to the device, wherein the working electrode includes glucose oxidase (and may also include catalase). The change in potential reflects the production of hydrogen peroxide, produced by the following reaction: ##STR1## The change in potential may be correlated to the concentration of glucose in the sample.
Riffer, U.S. Pat. No. 4,552,840 (issued Nov. 12, 1985), discloses an enzyme electrode for the analysis of dextran.
The electrode, from exterior to interior, comprises a sheath (cellulose sheath) including dextranase, a dialysis membrane (optional), a sheath including .alpha.-glucosidase, a sheath including glucose oxidase, and a platinum redox electrode. The electrode is immersed into a sample, containing an unknown amount of dextran and a known added amount of ferrocyanide (an excess). Dextran is hydrolyzed (by the enzymes in the various sheaths) to glucose. Glucose is oxidized to gluconic acid, and molecular oxygen is reduced to hydrogen peroxide. Ferrocyanide is oxidized by hydrogen peroxide and is used as an indicator of the peroxide generated. Potential changes as ferrocyanide is oxidized to ferricyanide. The change in potential is correlated to the concentration of dextran in the sample.
Nylander et al., U.S. Pat. No. 4,966,671 (issued Oct. 30, 1990), discloses a potentiometric analysis method, wherein a sample, containing an unknown amount of analyte (e.g., potassium), is added to each of two chambers. Each chamber includes an electrode (usually, similar electrodes, e.g., silver/silver chloride). The chambers are separated by a partition that permits ionic flow but is impermeable to the analyte being measured. One chamber includes a known amount of the analyte to be measured. When the sample to be measured is added to each chamber, the difference in concentration of analyte between the chambers creates a potential difference. The magnitude of the potential difference is a function of the amount of analyte in the sample.
None of these references discloses a simple potentiometric biosensor and assay method, wherein the analyte sought to be measured is anaerobically oxidized or reduced in an assay involving an enzyme, a redox mediator, and the analyte; and changes in system potential, which result from changes in the ratio of oxidized/reduced form of the redox mediator, are directly correlated to the detection or measurement of the analyte in the assay.